This application relates to detecting and quantifying substances in body fluids using fluorescence techniques.
Various methods for detecting and quantifying substances in body fluids are known. For example, in the case of glucose these methods include various colorimetric reactions, measuring a spectrophotometric change in the property of any number of products in a glycolytic cascade or measuring the oxidation of glucose using a polarographic glucose sensor.
U.S. Pat. No. 4,401,122 discloses an in vivo method for measuring substances such as glucose which involves placing an enzyme (e.g., glucose oxidase) either in or under the skin and detecting the enzymatic reaction product (e.g., oxygen) directly through the skin either colorimetrically or by using polarographic or enzyme electrodes. The amount of enzymatic reaction product detected is a measure of the amount of substrate.
Cerami, U.S. Pat. No. 4,330,299 describes an assay for measuring glucose concentration in which detection is carried out by means of color-producing agents or reagents which form precipitates. In one embodiment, for example, rhodamine is bonded to a lectin and a carbohydrate is affixed to a solid support. The rhodamine-labelled lectin reversibly binds to the carbohydrate in the absence of glucose. When glucose is introduced, it binds to the lectin, thereby displacing the rhodamine-labelled lectin from the carbohydrate. The complexed and uncomplexed rhodamine-lectin are then physically separated, and the reduction in color intensity displayed by rhodamine-lectin bound to carbohydrate is measured as an indication of glucose concentration.
Although such conventional assays have proven reliable, the reagents on which they rely become exhausted or must be removed. Therefore, these assays require the use of disposable sticks or replaceable cartridges, which can be expensive and inconvenient for the active user.
Meadows and Schultz describe another method by which blood glucose levels can be determined using optical means. They describe a fiber optic glucose sensor based on the competitive binding of glucose and fluorescein-labelled dextran (FITC-dextran) to rhodamine-labelled concanavalin A (Rh-Con A), Meadows, D. and J. S. Schultz, Talanta, 35:145-150 (1988).
The Meadows and Schultz optical sensor is attended by many problems, which means it is of limited use in a clinical setting or in monitoring blood glucose levels in individuals on a day to day basis. First, as mentioned in the article, the sensor can only detect glucose concentrations up to 2.00 mgs/ml. Although the normal blood glucose concentration in man is approximately 1.00 mg/ml., the concentration of glucose in diabetic blood can often exceed 3.00-4.00 mg/ml., which is well beyond the upper limit of the sensor described.
Second, Meadow's and Schultz's sensor has a short life because, as mentioned in the article, the dextran aggregates and becomes precipitated. Third, only 45% of the fluorescence is quenched using the Meadows and Schultz optical sensor. This effect may not be dramatic enough to be detected.
Finally, the in vivo use of a fiber optic is clinically impractical because in order to work, it must pierce the skin. Therefore, it requires an invasive technique and puts the patient at significant risk for developing serious infection. This is particularly true in diabetic patients who are known to have reduced resistance to infection.
An ideal sensor should be capable of detecting a wide range of physiological concentrations of analyte. As used herein, "physiological concentration" refers to the concentration of analyte found in both normal and pathological states. For example, in the case of glucose it refers to glucose levels found in normal, hypoglycemic, and hyperglycemic patients.
The sensor should also be reliable, reusable and easy to use. In addition, the in vivo sensor should be non-invasive.